The goal of this proposal is to characterize the effect of a large toxin from Gram negative bacteria and its effect on epithelial barrier function in vitro and in vivo. Lymphostatin (LS) is encoded by lymphocyte inhibitory factor A (lifA), a large gene present in Gram negative bacteria including Enterohemorrhagic, Enteropathogenic E. coli, and Citrobacter rodentium. lifA encodes for two critical enzymatic activities that have been implicated in microbial pathogenesis: a glycosyltransferase (1.6kb) and protease motif (4.5 - 4.8kb). We have generated two stable, specific in- frame insertion-deletion mutations inactivating both motifs. Our preliminary data suggest that LS is involved in disassembly of epithelial barrier function. It appears that the glycosyltransferase activity is responsible for the dissociation of ZO-1 from tight junctions by preventing activation of Cdc42. In addition, we provide evidence that the protease activity leads to dissociation of [unreadable]-catenin from adherens junctions by activating RhoA. Hypothesis 1: LS is critical for the disassembly of proteins forming the apical junctional complex (AJC), resulting in decreased transepithelial resistance (TER), promoting bacterial translocation and systemic dissemination. Aim 1: To study the effect of C. rodentium LS glycosyltransferase and protease activity on proteins constituting the AJC in differentiated epithelial cell cultures in vitro and in mice in vivo. Further, the effect of LS on members of the AJC appears to be mediated by causing an imbalance in Rho GTPase activation. We show that the protease motif regulates activation of Rho and suppresses Cdc42, whereas the glycosyltransferase is only involved in inhibition of Cdc42. Hypothesis 2: LS glycosyltransferase and protease activity are both critical for the disassembly of AJC components by regulating Rho GTPases. Aim 2: To investigate the activation cascade of Rho GTPases signaling in response to C. rodentium WT lifA, and inactivated protease- and glycosyltransferase motif in vitro and in vivo. The proposed research project will contribute to our understanding of how bacteria regulate epithelial barrier function during enteric infection and gain access to mucosa and submucosa with eventually systemic consequences. Lymphostatin is a large toxin from Gram negative bacteria, including pathogenic E. coli and Chlamydia spp. that account for significant morbidity and mortality by infecting gut, lungs, and the genitourinary tract. Our experimental results suggest that lymphostatin inhibits the immune response and regulates intestinal barrier function. The current proposal investigates the specific host proteins affected by lymphostatin and deregulated pathways.